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Russula albonigra is considered a well-known species, morphologically delimited by the context of the basidiomata blackening without intermediate reddening, and the menthol-cooling taste of the lamellae. It is supposed to have a broad ecological range and a large distribution area. A thorough molecular analysis based on four nuclear markers (ITS, LSU, RPB2 and TEF1-α) shows this traditional concept of R . albonigra s. lat. represents a species complex consisting of at least five European, three North American, and one Chinese species. Morphological study shows traditional characters used to delimit R . albonigra are not always reliable. Therefore, a new delimitation of the R . albonigra complex is proposed and a key to the described European species of R . subgen. Compactae is presented. A lectotype and an epitype are designated for R . albonigra and three new European species are described: R . ambusta , R . nigrifacta, and R . ustulata . Different thresholds of UNITE species hypotheses were tested against the taxonomic data. The distance threshold of 0.5% gives a perfect match to the phylogenetically defined species within the R. albonigra complex. Publicly available sequence data can contribute to species delimitation and increase our knowledge on ecology and distribution, but the pitfalls are short and low quality sequences.
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R E S E A R C H Open Access
Enlightening the black and white: species
delimitation and UNITE species hypothesis
testing in the Russula albonigra species
complex
Ruben De Lange
1*
, Slavomír Adamčík
2
, Katarína Adamčíkova
3
, Pieter Asselman
1
, Jan Borovička
4,5
,
Lynn Delgat
1,6
, Felix Hampe
1
and Annemieke Verbeken
1
ABSTRACT
Russula albonigra is considered a well-known species, morphologically delimited by the context of the basidiomata
blackening without intermediate reddening, and the menthol-cooling taste of the lamellae. It is supposed to have a broad
ecological range and a large distribution area. A thorough molecular analysis based on four nuclear markers (ITS, LSU, RPB2
and TEF1-α) shows this traditional concept of R.albonigra s. lat. represents a species complex consisting of at least five
European, three North American, and one Chinese species. Morphological study shows traditional characters used to delimit
R.albonigra are not always reliable. Therefore, a new delimitation of the R.albonigra complex is proposed and a key to the
described European species of R.subgen.Compactae is presented. A lectotype and an epitype are designated for R.
albonigra and three new European species are described: R.ambusta,R.nigrifacta, and R.ustulata. Different thresholds of
UNITE species hypotheses were tested against the taxonomic data. The distance threshold of 0.5% gives a perfect match to
the phylogenetically defined species within the R. albonigra complex. Publicly available sequence data can contribute to
species delimitation and increase our knowledge on ecology and distribution, but the pitfalls are short and low quality
sequences.
KEYWORDS: Basidiomycota, Coalescent species delimitation, Ectomycorrhizal fungi, New species, Phylogeny, Russulaceae,
Russulales,Russula subgen. Compactae, Integrative taxonomy, Typification, New taxa
INTRODUCTION
Molecular identification of species gained importance over
the last decades (Matute and Sepulveda 2019). As new tech-
niques became available and more easily accessible, the num-
ber of publications using sequence data increased immensely
(Hibbett et al. 2011; Kõljalg et al. 2013; Nilsson et al. 2018).
The main problems with molecular identification are poor
taxon coverage and misidentifications in many public
sequence databases, as well as high infraspecific variability of
DNA regions causing poor performance of the barcoding
gap (Kõljalg et al. 2005; Badotti et al. 2017;Hofstetteretal.
2019). To overcome some of these problems, the UNITE
database was created (https://unite.ut.ee/). UNITE targets the
still most widely used, universal fungal barcode: the nuclear
internal transcribed spacer (ITS) region to provide high-
quality reference records (Nilsson et al. 2018). UNITE groups
individual ITS sequences into species hypotheses (SHs) at
several distance thresholds (i.e. between 0 and 3%), each
assigned a unique digital object identifier (DOI) which allows
unambiguous reference across studies. These species hypoth-
eses are either assigned automatically with a representative
sequence or manually by a taxonomic expert with a
reference sequence (Kõljalg et al. 2013; Nilsson et al. 2018).
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* Correspondence: Ruben.DeLange@UGent.be
1
Research Group Mycology, Department of Biology, Ghent University, K.L.
Ledeganckstraat 35, 9000 Ghent, Belgium
Full list of author information is available at the end of the article
IM
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Fun
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De Lange et al. IMA Fungus (2021) 12:20
https://doi.org/10.1186/s43008-021-00064-0
Most Basidiomycota, the second largest phylum of
fungi, are Agaricomycotina (Naranjo-Ortiz and Gabaldon
2019). A considerable part of this subphylums diversity
is concentrated in large genera of ectomycorrhiza-
forming agarics, e.g. Cortinarius and Russula, which ex-
hibit very different evolutionary rates (Ryberg and
Matheny 2012; Varga et al. 2019). Recent studies on lin-
eages of closely related Russula members revealed that
they often comprise closely related species diversified by
ecological adaptation and isolation by distance or dis-
junction (Adamčík et al. 2016b; Caboňet al. 2019;
Looney et al. 2020). This makes the recognising of these
species relevant, despite their similarity in the ITS bar-
code. The efforts to barcode fungal species sometimes
fail, due to lack of taxonomic studies addressing the spe-
cies concept based on type collections (Kõljalg et al.
2020). Our current studies on Russula subgen. Compac-
tae recognised Russula albonigra as one such example.
Its morphological concept is historically established, and
the species is traditionally recognised by the moderately
distant, relatively narrow lamellae, the menthol-cooling
taste of the lamellae, and the surface of the cap and the
stipe, as well as the lamellae, that are strongly and rap-
idly blackening (hence, resulting in black-and-white con-
trast of bruised and untouched parts). Most publications
report blackening without intermediate reddening of the
context and surface (Romagnesi 1967; Adamčík and
Buyck 2014). Microscopically, the species is defined by a
low reticulate spore ornamentation and pileocystidia
without apical knobs (Romagnesi 1967). However, our
current search in two databases, which seek to adopt a
nomenclatural concept for fungal operational units de-
fined by the ITS barcode, recovered inconsistencies.
Samples identified as R. albonigra in the BOLD database
(https://www.boldsystems.org/) do not match any of the
species hypothesis under this species name published in
UNITE (https://unite.ut.ee/), and vice versa.
In this study, we use sequence data of four DNA
markers and a detailed morphological revision to test
the taxonomic status within the R. albonigra complex.
To test different distance thresholds of UNITE species
hypotheses, we used the strict genealogical concordance
assessing the extent of genetic concordance across loci,
the coalescent based species delimitation modelling the
genealogical history of individuals back to a common
ancestor and morphological differences.
MATERIAL AND METHODS
Sampling
This study is based on collections from sampling trips
in Belgium (2016, 2017 and 2018), Italy (1997, 2000
and 2016), Norway (2016), Slovakia (2003, 2006, 2008,
2009, 2011, 2015 and 2017), and Sweden (2016 and
2018). All collections are deposited in the Herbarium
Universitatis Gandavensis (GENT) or the Slovak
Academy of Sciences (SAV). Supplementary collec-
tions were requested from the Mycological Depart-
ment of the National Museum in Prague, Czech
Republic (PRM), and from the personal collections of
Felix Hampe, Jesko Kleine, and Helga Marxmüller
(the latter recently deposited in the State Museum of
Natural History Karlsruhe, KR).
Samples that could belong in the Russula albonigra
complex were selected based on morphology: (1) col-
lections that were identified as R.albonigra in the
field based on macro-morphology; and (2) fungarium
collections labelled as R.albonigra based on both
macro- and micro-morphological observations. The
selected samples were molecularly checked using the
ITS marker as a guideline and not included for
further study when not placed within the R.albonigra
complex.
Morphological analysis
The macroscopic description is based on observations
from fresh material, with colour codes following
Kornerup and Wanscher (1978), guaiac reactions refer-
ring to Chalange (2014), and spore print colour codes
following the scale of Romagnesi (1967). The micro-
scopic description and terminology follow Adamčík
et al. (2019). Microscopic characters were studied from
dried material, spores were observed in Melzers reagent,
elements of the hymenium and pileipellis were observed
in Congo red in L4 after ca. 10 s in KOH 10%. Basidio-
spores were measured using a crosshair eyepiece on a
Zeiss Axioskop 2 microscope. Line drawings of spores
were made based on stacked photographs (Nikon Eclipse
Ni-U microscope, stacking software: Extended Depth of
Field, Nikon Nis Elements module) at an original magni-
fication of 5000×. Measurements of other elements were
made using an eyepiece micrometer and line drawings
were prepared with the aid of a camera lucida (Olympus
U-DA) on an Olympus CX21 or BX43 microscope, at
original magnifications of 1500× or 2000×. Tissues were
mounted in Cresyl Blue (Buyck 1989), sulfovanillin
(Cabon et al. 2017) and treated with carbolfuchsin
(Romagnesi 1967) to observe the presence and colour
changes of incrustations and cystidium contents. All
cited collections in the species descriptions have been
sequenced, at least for ITS.
The key provided in the Taxonomy section is based on
our observations of species within the R.albonigra com-
plex, and following the traditional species concepts in
literature for the other taxa. Russula clementinae is not
included in the key as it is interpreted as a synonym of
R.densifolia (Sarnari 1998).
De Lange et al. IMA Fungus (2021) 12:20 Page 2 of 31
Molecular analysis
DNA extraction and amplification was performed in
either the molecular laboratory of Ghent University or
that of the Slovak Academy of Sciences. Sequencing of
PRM collections was conducted within the study of
Leonhardt et al. (2019).
In Ghent University, DNA from fresh material was ex-
tracted using the CTAB extraction described in Nuytinck
and Verbeken (2003). DNA from dried material was ex-
tracted using a modified CTAB protocol (Tel-Zur et al.
1999; modified by Meise Botanic Garden and Research
Group Mycology of Ghent University). The original proto-
col was optimized for cacti species. These plant types tend
to have larger cells compared to fungi. Hence, the ratio
DNA content:biomass for fungi is much higher. There-
fore, less biomass is needed as starting material and buffer
volumes were adjusted consequently. Because mucilagi-
nous polysaccharides have not been observed in previous
Fungal DNA extractions the use of sorbitol in our extrac-
tions was omitted. Additionally, the use of CTAB as deter-
gent to brake open fungal cells seemed to suffice to access
the fungal DNA in an efficient way. Hence, the extra sar-
kosyl added in the lysis step was also omitted from our
protocol. Protocols for PCR amplification follow Le et al.
(2007). In the Slovak Academy of Sciences, total genomic
DNA was extracted from dried material using the EZNA
Fungal DNA Mini Kit (Omega Bio-Tek, Norcross, GA,
USA) following the manufacturers instruction. Amplifica-
tion of DNA was performed in a PCR reaction mix con-
sisting of approximately 2 ng/μloftemplateDNA,forward
and reverse primers (10 pmol/μl), 5× HOT FIREPol® Blend
Master Mix (Solis BioDyne, Tartu, Estonia) and molecular
grade water added up to 20 μl. Four nuclear markers were
amplified: (1) the internal transcribed spacer region of
ribosomal DNA (ITS), comprising the ITS1 and ITS2 spa-
cer regions and the ribosomal gene 5.8S, using primers
ITS1-F and ITS4 (White et al. 1990; Gardes and Bruns
1993); (2) a part of the ribosomal large subunit 28S region
(LSU), using primers LR0R and LR5 (Moncalvo et al.
2000); (3) the region between the conserved domains 6
and 7 of the second largest subunit of the RNA polymer-
ase II (RPB2), using primers bRPB2-6F and fRPB27cR or
bRPB27.1R (Liu et al. 1999;Matheny2005); and (4) the
translation elongation factor 1-alpha (TEF1α), using pri-
mer pairs EF1-1018F and EF1-1620R or tef1F and tef1R
(Morehouse et al. 2003; Stielow et al. 2015). PCR products
from Ghent University were sequenced using an auto-
mated ABI 3730 XL capillary sequencer at Macrogen. In
the Slovak Academy of Sciences, the PCR products were
purified using Qiaquick PCR Purification Kit (Qiagen,
Hilden, Germany) and samples were sequenced by the
Seqme company (Dobříš, Czech Republic).
Forward and reverse sequences were assembled into
contigs and edited where needed with BioloMICS
(BioAware SA NV). All sequences generated were de-
posited in GenBank (Table 1).
Phylogenetic analysis
Identifications of publicly available sequences of fungi
often match contrasting taxonomic species concepts. To
provide reliable sampling in accordance with traditional
species concepts we selected three representative collec-
tions of each European species described within R. sub-
gen. Compactae, identified using the most recent and
reliable keys (Romagnesi 1967; Sarnari 1998). These col-
lections, together with the collections of R. albonigra s.
lat. were sequenced by us (Table 1). For non-European
species, sequences used in Adamčík et al. (2019)or
Buyck et al. (2020) were included if multiple of the
markers used in this study were available for these sam-
ples, with an ITS sequence obligatory. Four species of
Russula subgen. Archaeae were used as an outgroup, be-
cause the recent phylogenies of the genus place this sub-
genus as sister to R. subgen. Compactae.
Sequences were aligned using the online version of the
multiple sequence alignment program MAFFT v7
(Katoh and Toh 2008), using the E-INS-i strategy. Trail-
ing ends of the alignments were trimmed and the align-
ments were manually edited when necessary in MEGA7
(Kumar et al. 2016). The alignments can be obtained
from the first author and TreeBASE (Submission ID
26815). The alignments were partitioned into following
partitions: ITS-LSU-alignment: partial 18S, ITS1, 5.8S,
ITS2, LSU; RPB2-alignment: the RPB2 intron and the
first, second and third codon positions of the exon;
TEF1α-alignment: the first and second intron and the
first, second and third codon positions. PartitionFinder2
was used to find the appropriate partitioning scheme
and substitution models using the Akaike information
criterion (AICc) with a greedy search over all models
(Guindon et al. 2010; Lanfear et al. 2012; Lanfear et al.
2017). Maximum likelihood (ML) analyses were con-
ducted with IQ-Tree (Nguyen et al. 2014; Chernomor
et al. 2016) using standard bootstrapping analysis (1000
replicates). Bayesian inference (BI) was executed with
MrBayes v3.2.6 (Huelsenbeck and Ronquist 2001;
Ronquist and Huelsenbeck 2003). Two independent par-
allel runs of one cold and three heated chains were run
for ten million (single-locus datasets) or twenty million
generations (multi-locus dataset) with a sample fre-
quency of 100. Potential Scale Reduction Factor (PSRF)
values approached 1.0. Convergence and Effective Sam-
ple Size (ESS) statistics of the runs were also examined
with Tracer v1.7.1 (Rambaut et al. 2018). A burn-in sam-
ple of 20% was excluded before constructing the major-
ity rule consensus tree. Analyses were first performed on
each alignment separately and visually checked for in-
congruence. Significant incongruence was assumed if
De Lange et al. IMA Fungus (2021) 12:20 Page 3 of 31
Table 1 Specimens and GenBank accession numbers of DNA sequences used in the multi-locus phylogenetic analysis
Taxon Voucher collection (herbarium) Country ITS LSU RPB2 TEF1α
Russula acrifolia LD 16022 (GENT) Sweden MW172319 MW182479 MW306683 MW273325
Russula acrifolia RDL 18012 (GENT) Sweden MW172320 MW182480 MW306684 MW273326
Russula acrifolia RDL 18021 (GENT) Sweden MW172321 MW182481 MW273327
Russula adusta LD 16025 (GENT) Sweden MW172316 MW306682 MW273322
Russula adusta RDL 18020 (GENT) Sweden MW172317 MW182477 MW273323
Russula adusta RDL 18028 (GENT) Sweden MW172318 MW182478 MW273324
Russula albonigra JK RUS 13090603 (Jesko Kleine*) Germany MW172296 MW182461 MW306670
Russula albonigra SAV F-755 (SAV) Slovakia MW172291
Russula albonigra SAV F-2559 (SAV) Slovakia MW172292
Russula albonigra SAV F-20177 (SAV) Slovakia MW172298 MW182463 MW306672 MW273311
Russula albonigra SAV F-20197 (SAV) Slovakia MW172299 MW182464 MW306673 MW273312
Russula albonigra SAV F-3465 (SAV) Slovakia MW172293 MW182460 MW306669 MW273309
Russula albonigra SAV F-4776 (SAV) Slovakia MW172297 MW182462 MW306671 MW273310
Russula albonigra PRM 934322 (PRM) Czech Republic MW172294
Russula albonigra PRM 924409 (PRM) Czech Republic MW172295
Russula ambusta FH 2008 ST01 (Felix Hampe*) Germany MW172300 MW182465
Russula ambusta SAV F-3358 (SAV) Slovakia MW172301 MW182466
Russula cf. anthracina RDL 16031 (GENT) Italy MW172313 MW182474 MW306679 MW273319
Russula cf. anthracina RDL 16058 (GENT) Italy MW172314 MW182475 MW306680 MW273320
Russula cf. anthracina RDL 18026 (GENT) Sweden MW172315 MW182476 MW306681 MW273321
Russula archaeosuberis BB 12.085 (PC) Italy KY800355° KU237593° KU237878° KU238019°
Russula atramentosa FH 2011-002R (Felix Hampe*) Belgium MW172322 MW182482 MW306685 MW273328
Russula atramentosa RDL 16050 (GENT) Italy MW172323 MW306686
Russula atramentosa FH017082402 (Felix Hampe*) Germany MW172324 MW182483 MW306687 MW273329
Russula camarophylla MPG117-09 (PC) Spain KY800353° KU237579° KU237865° KU238006°
Russula cantharellicola UC1999420 United States KF306036°
Russula cascadensis OSC 1064009 (OSC) United States EU526006°
Russula cortinarioides BB 07.133 (PC) United States KP033485° KP033507°
Russula cortinarioides BB 07.103 (PC) United States KP033480° KP033491° KP033502° KU237985°
Russula densifolia RDL 16001/2 (GENT) Belgium MW172325 MW182484 MW306688 MW273330
Russula densifolia RDL 18052 (GENT) Belgium MW172326 MW182485 MW306689 MW273331
Russula densifolia RDL 17024 (GENT) Belgium MW172327 MW182486 MW306690 MW273332
Russula densissima FH 2014 ST04 (Felix Hampe*) Germany MW172328 MW306691
Russula densissima FH 2010 ST02 (Felix Hampe*) Germany MW172329 MW306692
Russula dissimulans BPL704 (TENN) United States KY848513°
Russula earlei BPL245 (TENN) United States KT933961° KT933820° KT933891°
Russula cf. eccentrica BB 07.044 (PC) United States KP033479° KP033490° KP033501° KU237937°
Russula cf. eccentrica BB 07.132 (PC) United States KP033478° KP033489° KP033500° KU237926°
Russula cf. fuliginosa FH RUS 14091001 (Felix Hampe*) Slovakia MW172330 MW182487 MW306693 MW273333
Russula cf. fuliginosa FH RUS 14091201 (Felix Hampe*) Slovakia MW172331 MW182488 MW306694 MW273334
Russula gossypina BB 06.002 (PC) Madagascar KY800350° KU237450° KU237736° KU237886°
Russula ingwa MEL2101936 Australia EU019919°
Russula khanchanjungae AV KD KVP 09106 (GENT) India KR364129° JN389004° JN375607°
Russula lateriticola BB 06.031 (PC) Madagascar KP033476° KP033487° KP033498° KU237888°
De Lange et al. IMA Fungus (2021) 12:20 Page 4 of 31
two different relationships (one monophyletic and the
other non-monophyletic) for any set of taxa were sup-
ported with bootstrap values (BS) 70 or posterior prob-
abilities (PP) 90. The resulting gene trees did not show
any supported conflicts, therefore all alignments could
be concatenated. The concatenated alignment was used
for the multi-locus phylogenetic analyses (Fig. 1).
Coalescent species delimitation approaches
For species delimitation under the multispecies coales-
cent model a part of the alignment used in the multi-
locus phylogenetic analyses mentioned above, compris-
ing members of the Russula albonigra complex, was
used. A total of five potential species units (as proposed
by the ML and BI trees) were evaluated as the full
model. Two coalescent species delimitation methods
were performed to test these species hypotheses. The
first method was implemented in Bayesian Phylogenetics
and Phylogeography, BP&P v4.3.8 (Yang 2015). We per-
formed analysis A11 (Yang and Rannala 2014) for un-
guided species delimitation using rjMCMC algorithm 0
(Yang and Rannala 2010). Analyses were run with sev-
eral values for the fine-tune parameter (ε= 2, 5, 10 and
20) and we assigned equal probabilities to the rooted
species trees as a species model prior. Because the prior
distributions on the ancestral population size and root
age can affect the posterior probabilities of the model,
we considered three different combinations of priors
(based on the idea of Leache and Fujita (2010)): (1) θ~
IG(3,0.002) and τ~ IG(3,0.002), (2) θ~ IG(3,0.02) and
τ~ IG(3,0.02), and (3) θ~ IG(3,0.02) and τ~ IG(3,0.002)
(with α= 3 for a diffuse prior as proposed in the man-
ual). For each combination of settings the analysis was
run twice with a different seed (to confirm consistency
between runs) for 200,000 generations (sampling interval
of two) and a burn-in of 50,000. As a second species de-
limitation method we used the STACEY v1.2.5 (Jones
2017) package implemented in BEAST2 (Bouckaert et al.
2019). The xml-file for the BEAST2 runs were prepared
in BEAUTi v2.6.3 (Drummond et al. 2012). We used fol-
lowing partitions: for the nrDNA (1) 5.8S, (2) ITS1 +
ITS2 and (3) LSU; for the protein coding loci the introns
and the first, second and third codon positions of the
exons. PartitionFinder2 was used to find the appropriate
substitution models. The substitution rate of each parti-
tion was estimated independently of the others. Clock
and tree model parameters were estimated independ-
ently for the nrDNA and each protein coding locus. We
used a lognormal, relaxed clock model and a Yule tree
model. The Collapse Height parameter εwas set to
10
5
. The Collapse Weight parameter ωwas estimated
and given a uniform prior on [0,1] so that every number
of species between five and one is regarded as equally
likely a priori. We ran five parallel MCMC runs for one
Table 1 Specimens and GenBank accession numbers of DNA sequences used in the multi-locus phylogenetic analysis (Continued)
Taxon Voucher collection (herbarium) Country ITS LSU RPB2 TEF1α
Russula nigricans RDL 17004 (GENT) Belgium MW172332 MW182489 MW306695 MW273335
Russula nigricans RDL 17007 (GENT) Belgium MW172334 MW182491 MW306697 MW273337
Russula cf. nigricans RDL 17005 (GENT) Belgium MW172333 MW182490 MW306696 MW273336
Russula nigrifacta RDL 16028 (GENT) Italy MW172307 MW306676 MW273316
Russula nigrifacta RDL 16044 (GENT) Italy MW172308 MW182470 MW306677 MW273317
Russula nigrifacta RDL 16063 (GENT) Italy MW172306 MW273315
Russula nigrifacta SAV F-2418 (SAV) Slovakia MW172304
Russula nigrifacta SAV F-2419 (SAV) Slovakia MW172303 MW182468
Russula nigrifacta SAV F-1501 (SAV) Slovakia MW172302 MW182467 MW306674 MW273314
Russula nigrifacta SAV F-3006 (SAV) Slovakia MW172305 MW182469 MW306675
Russula polyphylla BB 07.134 (PC) United States KP033486° KP033497° KP033508° KU238023°
Russula polyphylla BB 07.023 (PC) United States KP033481° KP033492° KP033503° KU237986°
Russula roseonigra KR-M-0042973/MxM R-9308 (KR) France MW172335
Russula roseonigra FH 2014 ST01 (Felix Hampe*) Germany MW172336 MW306698 MW273338
Russula roseonigra RDL 16024 (GENT) Italy MW172337 MW182492 MW306699 MW273339
Russula sp. FH 12064 (GENT) Thailand MN130076° MN380517°
Russula sp. 1 RW 1975 (GENT) Italy MW172309 MW182471
Russula ustulata AV 16019 (GENT) Norway MW172312 MW182473 MW306678 MW273318
Russula ustulata SAV F-2610 (SAV) Italy MW172310 MW182472
Russula ustulata PRM 924452 (PRM) Czech Republic MW172311
In bold: types; * personal herbarium; ° sequences not generated in this study
De Lange et al. IMA Fungus (2021) 12:20 Page 5 of 31
Fig. 1 Maximum Likelihood (ML) tree of Russula subgen. Compactae, based on concatenated ITS, LSU, RPB2 and TEF1αsequence data. ML bootstrap values > 75 and BI posterior probabilities > 0.95
are shown
De Lange et al. IMA Fungus (2021) 12:20 Page 6 of 31
0.03
KX441086 China
UDB0641772 Estonia
JF519253 Austria
UDB038057 NOBAS948-15 O-F-249610 Norway
KF306042 United States
JF908707 Italy
KF306043 United States
JF519235 Austria
JF519171 Austria
UDB0367026 Estonia
JF519248 Austria
UDB0392273 Estonia
KT800130 United States
UDB0448007 Estonia
DQ422029 Sweden
UDB0669827 Estonia
HM488592 United States
KJ769287 Russian Federation
UDB031523 Estonia
UDB0513905 Estonia
UDB011193 Estonia
NOBAS1861-16 O-F-251464 Norway
UDB051783 Estonia
UDB0287633 Estonia
JF519228 Austria
KF306041 United States
KF306040 United States
NOBAS1778-16 O-F-251577 Norway
UDB051911 Estonia
UDB063604 Estonia
NOBAS1869-16 O-F-251473 Norway
JF834364 United States
UDB0661958 Estonia
JF834355 United States
UDB0211768 Estonia
UDB0703616 Estonia
UDB031522 Estonia
UDB0660450 Estonia
NOBAS4607-17 O-F-253969 Norway
UDB0299885 Estonia
UDB0701696 Estonia
UDB0585705 Estonia
UDB0459908 Estonia
FJ013070 Spain
UDB0536051 Estonia
UDB016040 Finland
AB291763 Japan
UDB037058 NOBAS3604-16 O-F-26206 Norway
UDB0602964 Estonia
UDB024525 Laos
UDB011240 Estonia
UDB0273348 Estonia
UDB0703036 Estonia
95
92
100
99 100
100
100
100
96
100
96
100
100
99
75
99
93
88
100
99
100
82
90
98
99
100
71
100
100
77
99
79
64
63
100
94
65
69
//
0.5% SH1961382.08FU
<0.5% SH2310095.08FU
1% SH1740694.08FU
1.5% SH1569600.08FU
2% SH1425818.08FU
2.5% SH1300493.08FU
3% SH1188805.08FU
<0.5% SH2310133.08FU
0.5% SH1961409.08FU
<0.5% SH2310129.08FU
<0.5% SH2310126.08FU
0.5% SH1961425.08FU
0.5% SH1961426.08FU
<0.5% SH2310140.08FU
0.5% SH2890350.08FU
1% SH1740706.08FU
1.5% SH1569600.08FU
2% SH1425829.08FU
2.5% SH1300506.08FU
3% SH1188820.08FU
0.5% SH1961392.08FU
<0.5% SH3011319.08FU
<0.5% SH2310110.08FU
MH930188 Russian Federation
UDB065518 Estonia
UDB0557800 Estonia
UDB0502905 Estonia
UDB0663165 Estonia
representative sequence locked
R. albonigra R. ustulata R. ambusta R. nigrifacta
<.5
0.5
1.0
1.5
2.5
2.0
3.0
%
R. albonigra lineage
66
0.5% SH1961425.08FU
Russula nigrifacta SAV F-3006 Slovakia
Russula nigrifacta SAV F-1501 Slovakia
Russula nigrifacta RDL 16-028 Italy
Russula nigrifacta RDL 16-044 Italy HOLOTYPE
Russula nigrifacta SAV F-2418 Slovakia
Russula nigrifacta SAV F-2419 Slovakia
Russula nigrifacta RDL 16-063 Italy
Russula ambusta FH 2008 ST01 Germany
Russula ambusta SAV F-3358 Slovakia HOLOTYPE
Russula ustulata AV 16-019 Norway HOLOTYPE
Russula ustulata PRM 924452 Czech Republic
Russula ustulata SAV F-2610 Italy
Russula albonigra SAV F-20177 Slovakia
Russula albonigra JK RUS 13090603 Germany
Russula albonigra SAV F-3465 Slovakia
Russula albonigra SAV F-4776 Slovakia
Russula albonigra SAV F-755 Slovakia
Russula albonigra PRM 934322 Czech Republic
Russula albonigra SAV F-20197 Slovakia EPITYPE
Russula albonigra SAV F-2559 Slovakia
Russula albonigra PRM 924409 Czech Republic
Russula sp. 1 RW 1975 Italy
Russula roseonigra KR-M-00242973/MxM R-9308 France
Russula roseonigra RDL 16-024 Italy
Russula roseonigra FH 2014 ST01 Germany
Russula cf. anthracina RDL 16-031 Italy
Russula cf. anthracina RDL 18-026 Sweden
Russula cf. anthracina RDL 16-058 Italy
Russula acrifolia LD 16-022 Sweden
Russula acrifolia RDL 18-012 Sweden
Russula acrifolia RDL 18-021 Sweden
Russula cascadensis EU526006 United States
Russula adusta LD 16-025 Sweden
Russula adusta RDL 18-028 Sweden
Russula adusta RDL 18-020 Sweden Russulaaff. densifolia
Russula aff. densifolia AB291759 Japan
Russula aff. densifolia AB291758 Japan
AB291755 JapanRussula aff. densifolia
Russula sp. FH 12-064 Thailand
Russula atramentosa FH 2011-002R Belgium
Russula atramentosa RDL 16-050 Italy
Russula atramentosa FH0170824-02 Germany
Russula aff. densifolia AB291762 Japan
Russula aff. densifolia AB291756 Japan
Russula densissima FH 2014 ST04 Germany
Russula densissima FH 2010 ST02 Germany
Russula densifolia RDL 16-001/2 Belgium
Russula densifolia RDL 17-024 Belgium
Russula densifolia RDL 18-052 Belgium
Russula aff. densifolia AB291761 Japan
Russula aff. densifolia AB291760 Japan
Russula aff. densifolia AB291757 Japan
Russula cf. fuliginosa FH RUS 14091001 Slovakia
Russula cf. fuliginosa FH RUS 14091201 Slovakia
Russula dissimulans KY848513 United States
Russula nigricans RDL 17-004 Belgium
Russula nigricans RDL 17-007 Belgium
Russula cf. nigricans RDL 17-005 Belgium
Fig. 2 Maximum Likelihood (ML) tree of Russula sect. Nigricantinae, based on ITS sequence data. ML bootstrap values > 60 are shown. Colour bars of
similar colour within each column represent individual UNITE species hypotheses. Missing bars represent unresolved clustering. Samples in colour are
representative sequences labelled with SH numbers. Grey shaded samples in white are plotted to the tree based on similarities in distinguishing
nucleotide positions (uncertain positions are labelled by grey dotted lines)
De Lange et al. IMA Fungus (2021) 12:20 Page 7 of 31
billion generations sampling every 1000th tree. Conver-
gence and Effective Sample Size (ESS) statistics of the
runs were examined with Tracer v1.7.1. Twenty percent
of each run was discarded as burn-in and the remaining
posterior samples were combined using LogCombiner
v2.6.3 (Drummond and Rambaut 2007) and used to cal-
culate the most likely number of clusters (i.e., putative
species), using SpeciesDelimitationAnalyzer (Jones et al.
2014).
Species hypothesis and threshold testing
ITS sequences generated by authors of this study and
used in the multi-locus phylogeny were combined with
all ITS sequences, either labelled as R.albonigra or
showing high similarity (97%) to our sequences of
R. albonigra s.lat., available on UNITE (https://unite.ut.ee/),
GenBank (www.ncbi.nlm.nih.gov)andBOLD(https://www.
boldsystems.org/) databases. Accession numbers are given
in Fig. 2. The clade containing R. nigricans and R. dissimu-
lans is in a basal position within R.sect.Nigricantinae (see
Fig. 1) and is chosen as the outgroup. Short sequences
(containing only ITS1 or ITS2) and sample UDB065518
(containing many ambiguities and differences in conserved
domains compared to other sequences of the group)
were excluded from the analysis. The sequences were
aligned following the same principles as mentioned
above. The alignment was partitioned into following
partitions: ITS1, 5.8S and ITS2. A ML analysis was con-
ducted with IQ-Tree (Nguyen et al. 2014;Chernomor
et al. 2016) using the option to first test for the best
substitution model (Kalyaanamoorthy et al. 2017)and
standard bootstrapping analysis (1000 replicates). The
excluded samples were later plotted on the tree based
on similarities in distinguishing nucleotide positions
(Additional file 1). We identified the single nucleotide
positions distinguishing the species of the R.albonigra
complex and compared the excluded samples to the
good quality sequences based on these positions to esti-
mate their placement in the tree. SH inclusiveness across
sequence distance threshold values, as it is shown on
UNITE, is plotted against the tree.
RESULTS
Multi-locus phylogenetic analyses
All 24 sequences generated by the authors of specimens
putatively identified as R. albonigra, are placed within R.
subgen. Compactae and group together with other mem-
bers of R. sect. Nigricantinae (Fig. 1). All but two of
these sequences are placed in one strongly supported
clade, here further referred to as the R.albonigra com-
plex. The two sequences outside the R.albonigra com-
plex are placed in either the R.atramentosa clade or the
R. anthracina clade (these sequences were not used for
the final analysis and are not shown in the trees). Our
molecular analysis shows the presence of five distinct
European clusters within the R.albonigra complex (Fig.
1). They form four well supported clades and one single-
ton collection on a long branch. The overall topology of
the ML and BI tree was congruent.
The name R.albonigra is assigned to a clade with two
collections from the Czech Republic, PRM 924409
and PRM 934322, that originate from the type collecting
area. Three species, R.ambusta,R.nigrifacta and R.ustulata,
are described here as new. One species is represented by a
singleton position in the tree, placed as sister to R. albonigra
and is labelled as Russula sp. 1. These two species
form a sister clade to a larger clade containing R.ambusta,
R.nigrifacta, and R.ustulata. The relations within the
latter clade are not well supported in the BI tree.
Coalescent-based species delimitation
The full set of proposed species (i.e. five species) was re-
covered as the highest supported species model in the
BP&P analysis under each combination of settings, with
posterior probabilities ranging from 0.91 to 0.99. Also
the STACEY analysis resulted in the highest probability
(posterior probability of 0.99) for five minimal clusters
(species). Both coalescent delimitation methods confirmed
the species hypothesis for all five clusters in the multi-
locus phylogenetic analysis.
ITS analysis and optimal SH distance
When searching for UNITE species hypotheses labelled
as R. albonigra, at every threshold, two species hypoth-
eses are found that are not placed within the R.alboni-
gra complex defined by our multi-locus analysis. Both
are represented by a singleton sequence. The first,
UDB024525 represents a collection from Lao Peoples
Democratic Republic and seems more closely related to
R. atramentosa. The second, JF908707 represents a
collection from Italy with an isolated position in the
phylogeny (Fig. 2).
The general topology of the ITS tree is congruent with
the multi-locus tree and all sequences of the R. albonigra
complex generated by the authors of this study are again
placed within this monophyletic group (Fig. 2). The ITS
analyses revealed the presence of three additional North
American clusters and one Chinese collection of singleton
position, within the R. albonigra complex. Two of the
North American clusters are supported and probably rep-
resent well defined species, while the status of the three
sequences from the USA (KF306041, JF834355 and
KF306040) is uncertain and requires more sequence data
to resolve. The singleton Chinese sample (KX441086)
probably represents an undescribed species sister to R.
albonigra. Furthermore, Fig. 2shows an overview of the
different UNITE species hypotheses within the R.alboni-
gra complex at different thresholds. At a threshold of 1%
De Lange et al. IMA Fungus (2021) 12:20 Page 8 of 31
or higher two species hypotheses are recognised (for SH
numbers see red and pink boxes in the Fig. 2). The first
one labelled as R. albonigra with the representative
UNITE sequence UDB016040 covers R.ambusta,R.nigri-
facta,R.ustulata and the North American species. The
second one with the representative sequence JF519228 is
labelled as Russula sp.and covers R.albonigra and the
Chinese species. At a threshold of 0.5%, all European spe-
cies (except for R. sp. 1 which is not represented by any
public sequence) and two North American species are
supported. Threshold < 0.5% gives additional units within
the phylogenetic species that were not supported by our
multi-locus analysis.
TAXONOMY
The species within the Russula albonigra complex are
characterised by the moderately distant, relatively nar-
row lamellae, the context that is rapidly and strongly
blackening, generally without intermediate reddening. In
some cases though, some slight reddening is observed.
The taste of the lamellae and flesh is never acrid, but
can be menthol-cooling. Microscopically, the species are
Fig. 3 Basidiomata. a-b Russula nigrifacta (RDL 16044, holotype). c-f Russula albonigra (c: SAV F-20197, epitype; dPRM 934322; ePRM
924409; fSAV F-20177). gRussula ambusta (FH 2008 ST01). hRussula ustulata (AV 16019, holotype)
De Lange et al. IMA Fungus (2021) 12:20 Page 9 of 31
defined by spores with low and dense warts forming
subreticulate to reticulate ornamentation, long pileocys-
tidia (if present) and a cystidial content which is not
reacting in sulfovanillin.
Russula albonigra (Krombh.) Fr., Hymenomyc.eur.
(Upsaliae): 440 (1874). (Figs. 3c-f, 4,5and 6).
Basionym:Agaricus alboniger Krombh., Naturgetr.
Abbild. Beschr. Schwämme (Prague) 9: 27 (1845).
Type: Krombholz (1845)lectotype designated here,
MB10000343); Slovakia: Oblík Nature reserve, with
Fagus, 23 Sept. 2017, S. Adamčík (SAV F-20179 epi-
type designated here, MB10000342).
Description:Pileus large, 56112 mm diam., plano-
convex, at the centre with shallow but wide depression;
margin deflexed, long involuted, not striated, smooth; pi-
leus surface velvety and smooth near margin, towards
the centre radially wrinkled or rugulose, centre smooth,
dry, matt, almost not peeling (max. to 1/3 of the radius);
young completely white, later becoming yellowish white
(4A2), cream (4A3) to orange-grey (5B2) at the centre,
more greying and blackening when old. Lamellae seg-
mentiform to subventricose, to 6 mm deep, adnate to
subdecurrent; snow white, later yellowish white (4A2),
blackening with age or when bruised; with numerous
lamellulae of different lengths, frequently forked near
Fig. 4 Russula albonigra (SAV F-755, SAV F-2559, SAV F-20177), hymenium. aBasidia. bMarginal cells. cBasidiospores. dCystidia near lamellae
edges. eCystidia on lamellae sides. Bar = 10 μm, except for c 5 μm
De Lange et al. IMA Fungus (2021) 12:20 Page 10 of 31
the stipe but also near the pileus margin, often anas-
tomosed; moderately crowded to moderately distant,
L = 190260, l = 1 (one between each pair of long
lamellae); edges even, concolorous, blackening with age.
Stipe 4260 × 1429mm, cylindrical, firm and fleshy, lon-
gitudinally striated, velvety near the lamellae; white, later
becoming greyish orange (5B4) near the base; interior
solid, cortex ca. 2.5 mm thick. Context ca. 7 mm thick at
mid-radius, hard, white, turns rapidly grey and then black
on cut section, at surface also turns red before grey and
black; turning orange with FeSO
4
, immediately dark blue
with guaiac (strong reaction, +++); taste mild, slightly like
mint (refreshing) in lamellae, odour weak of apples. Spore
print white (Ia).
Basidiospores (7.1)7.58.08.5(9.4) × (5.6)5.96.36.7(
7.1) μm, broadly ellipsoid to ellipsoid, Q = (1.16)1.221.29
1.36(1.49); ornamentation of low, dense [(6)710(11) in a
3μm diam circle] amyloid warts, 0.10.4 μm high, subreticu-
late, abundantly fused into chains [(0)37(8) fusions in a
3μm diam circle], and also connected by short, fine line
Fig. 5 Russula albonigra (SAF F-755, SAV F-20177), hyphal terminations of the pileipellis. aNear the pileus margin. bNear the pileus centre. Bar = 10 μm
De Lange et al. IMA Fungus (2021) 12:20 Page 11 of 31
connections [04(6) in a 3 μm diam circle]; suprahilar spot
medium-sized, not amyloid. Basidia (44)48.955.1
61.3(75) × 10.010.811.6(13) μm, narrowly clavate,
4-spored. Hymenial cystidia (65)69.685.8
102.0(115) × (7)7.68.59.4(10) μm, cylindrical to
narrowly fusiform, apically obtuse to mucronate, thin-
walled; with little content composed of large pale, oily, re-
fringent guttules, without reaction in sulfovanillin; near
the lamellae edges, (30)40.458.576.6(98) × (6)7.1
7.98.7(9) μm, cylindrical to narrowly fusiform, some-
times slightly flexuose, apically obtuse to mucronate or
with small appendage, thin-walled, content as on lamellae
sides. Lamellae edges sterile, when older elements can
contain brown pigments; marginal cells (11)16.522.1
27.7(31) × (3)3.54.96.3(8) μm, poorly differenti-
ated, cylindrical, flexuose, thin-walled. Pileipellis ortho-
chromatic in Cresyl Blue, 8090 μm deep, not sharply
delimited from trama and not gradually passing, inter-
mediate; subpellis not delimited from suprapellis; hyphae
36μm wide near trama, not regular in width, dense,
homogeneous, pigmented only near the surface, with no
distinct gelatinous coating or only weakly on deeper hy-
phae. Acid-resistant incrustations absent. Hyphal termina-
tions near the pileus margin long, with multiple septa,
flexuous, thin-walled, filled with irregular refractive bodies
containing brown pigments; terminal cells very long
(35)55.986.6117.3(160) × (5)5.26.68.0(10) μm,
narrowly cylindrical to subulate, on average apically con-
stricted to 3.5 μm (average difference of 3.2 μmbetween
maximum width and width of the tips); subterminal cells
and the cells below usually shorter and gradually wider,
subterminal cells occasionally branched. Hyphal termina-
tions near the pileus centre slightly slender and apically
less attenuated; terminal cells slightly shorter (40)51.8
72.994.0(124) × (4)4.56.07.5(10) μm, subterminal
cells never branched. Pileocystidia near the pileus margin
widely dispersed, 13 celled, long, terminal cells
(61)73.097.5122.0(160) × (5)5.57.08.5(10) μm,
cylindrical to subulate, flexuose, sometimes with small
lateral projection, apically obtuse or with 12 eccen-
tric appendages, sometimes bifurcating, with oily gut-
tulate content, without reaction in sulfovanillin; near
the pileus centre widely dispersed, 12 celled, gener-
ally shorter, terminal cells (52)58.075.292.4(
115) × (4)5.16.88.5(11) μm, similar in shape and
content, mostly apically with 12 eccentric append-
ages, not bifurcating. Pileocystidia not near surface
but only deeper in the pileipellis. Oleiferous hyphae
containing brown pigments and cystidioid hyphae
present in the trama.
Ecology: Growing with Fagus sylvatica,Abies alba,
Picea abies, and Carpinus betulus.
Distribution: Known from Austria, the Czech Republic,
Estonia, Germany, Norway, and Slovakia.
Fig. 6 Russula albonigra (SAV F-20177), pileocystidia. aNear the
pileus margin. bNear the pileus centre. Bar = 10 μm
De Lange et al. IMA Fungus (2021) 12:20 Page 12 of 31
Additional material studied:Slovakia:Stužica Natural
reserve, under Kýčera hill, with Abies and Fagus, 22 Sept.
2017, S. Adamčík, (SAV F-20177); Stužica National Nature
Reserve, central part, with Fagus sylvatica, 5 Oct. 2003, S.
Adamčík, (SAV F-755); Badínsky prales Nature reserve,
with Abies and Fagus, 22 Sept. 2017, S. Adamčík,(SAVF-
2559); Badínsky prales Nature reserve, with Abies and
Fagus, 29 Sept. 2011, S. Adamčík, (SAV F-3465); Revúca,
road to Sirk, with Fagus sylvatica, 14 Oct. 2015, S. Adam-
čík, (SAV F-4776). Germany: Bavaria, Oberallgäu, Ober-
staufen, Hündle, alt. 975 m, N47°3245.9E10°0415.1,
with Abies alba,Fagus sylvatica and Picea abies, 6 Sept.
2013, J. Kleine, JK RUS 13090603 (hb. Jesko Kleine).
Czech Republic: Central Bohemia, Chrudim District,
Bojanov (Horní Bezděkov), under Fagus, 12 Aug. 2012, J.
Borovička, (PRM 934322); Central Bohemia, Kladno
District, Běleč(Jenčov), under Fagus and Carpinus,3Sept.
2013, J. Borovička, (PRM 924409).
Notes:Russula albonigra was first described by
Krombholz (1845)asAgaricus alboniger with only a
brief description and no holotype designated. Later, Fries
classified it in the genus Russula under its current name
(Fries 1874) that became well-known and widely used in
Europe. The illustration (Krombholz 1845: pl. 70, Figs. 16
and 17; Biodiversity Heritage Library), reproduced in
Fig. 7and made by Krombholz was suggested to serve as
lectotype by Sarnari, although it was never formally des-
ignated (Sarnari 1998). The illustration is the only avail-
able original material and is hereby formally designated
as the lectotype. The brief description and the plate itself
are not sufficient to determine which species of the
R. albonigra complex corresponds to R.albonigra.
This makes the collecting area of Krombholz the
most relevant criterion and in Krombholz (1845)itis
mentioned that R.albonigra is found in Prague.
Within the dataset, collection PRM 924409 was found
Fig. 7 Krombholz (1845). Image from the BHL (Biodiversity Heritage Library). Contributed by Missouri Botanical Garden, Peter H. Raven Library
De Lange et al. IMA Fungus (2021) 12:20 Page 13 of 31
only 31 km from the Prague city centre. Therefore,
the clade in which this collection is placed, is chosen
to represent R.albonigra. Specimen SAV F-20197 is
here designated as epitype.
Russula ambusta De Lange, Adamčík & F. Hampe,
sp. nov. (Figs. 3g, 8,9and 10).
MycoBank: MB839080.
Etymology: Refers to the appearance of the basidio-
mata, which look like they were burnt.
Diagnosis: Differs from the other species of the
Russula albonigra complex by the intermediate
reticulation and density of spore ornamentation and
the presence of appendages, but lack of bifurcations
on the pileocystidia.
Type:Slovakia: Vývrať,Bučková, W slopes of the hill,
with Quercus, 6 July 2011, V. Kučera (SAV F-3558
holotype).
Description: Pileus large, 45100 mm diam, planocon-
vex to applanate, centrally depressed to umbilicate,
becoming more infundibuliform when older; margin
slightly inflexed when young, straight when mature,
smooth; pileus surface smooth, dry, dull to somewhat
viscid when wet; greyish orange, light brown (5B5, 5D5)
Fig. 8 Russula ambusta (SAV F-3358, FH 2008 ST01), hymenium. aBasidia. bMarginal cells. cBasidiospores. dCystidia near lamellae edges.
eCystidia on lamellae sides. Bar = 10 μm, except for c 5 μm
De Lange et al. IMA Fungus (2021) 12:20 Page 14 of 31
to umber, greyish brown (5F8, 6F3) with paler white to
sand coloured patches. Lamellae segmentiform to sub-
ventricose, to 6 mm deep, adnate to subdecurrent; snow
white, later yellowish white (4A2), blackening with age
or when bruised; with numerous lamellulae of different
lengths; dense (710 L + 58 l/cm at mid-radius); edges
even, concolorous, blackening with age. Stipe 3040 ×
1525 mm, cylindrical, firm and fleshy, smooth; white
but rapidly almost completely orange brown; solid in-
side. Context ca. 35 mm thick at mid-radius, firm,
white, greying before blackening, no reddening observed;
turning immediately dark blue with guaiac (strong reac-
tion, +++); taste first mild, then quickly somewhat cool-
ing with menthol component, never spicy or acrid;
odour indistinct. Spore print white (Ia).
Fig. 9 Russula ambusta (SAV F-3358, FH 2008 ST01), hyphal
terminations of the pileipellis. aNear the pileus margin. bNear the
pileus centre. Bar = 10 μm
Fig. 10 Russula ambusta (SAV F-3358), pileocystidia. aNear the
pileus margin. bNear the pileus centre. Bar = 10 μm
De Lange et al. IMA Fungus (2021) 12:20 Page 15 of 31
Basidiospores (6.7)7.48.29.0(9.6) × (5.4)5.76.0
6.3(6.8) μm, broadly ellipsoid to narrowly ellipsoid, Q =
(1.18)1.281.381.48(1.54); ornamentation of very low,
dense to very dense [(6)713(16) in a 3 μmdiamcircle]
amyloid warts, up to 0.2 μm high, subreticulate to reticulate,
abundantly fused in chains [(2)38(10) fusions in a 3 μm
diam circle], and also connected by short, fine line connec-
tions [(0)19(17) in a 3 μm diam circle]; suprahilar spot
medium-sized, not amyloid. Basidia (54)57.562.667.7(
76) × (8)8.99.510.1(11) μm, narrowly clavate, 4-spored.
Hymenial cystidia (66)67.191.9107.7(125) × (7)8.5
9.811.1(13) μm, variable: (1) narrowly fusiform to
narrowly clavate, flexuose to even slightly moniliform,
apically obtuse or with a constriction or with small
appendage to even slightly mucronate, thin-walled;
with heteromorphous, oily content, mostly fragmented
in multiple crystalline-like masses or slightly granu-
lose, without reaction in sulfovanillin; (2) narrowly
fusiform to lanceolate, flexuose, tapering towards the
top; with less content, heteromorphous, oily, mostly
fragmented in multiple crystalline-like masses or
slightly granulose, without reaction in sulfovanillin; near
the lamellae edges, (29)37.063.790.4(144) × (5)7.2
8.59.8(10) μm, (1) narrowly fusiform to narrowly cla-
vate, flexuose, apically obtuse or tapering towards the top
in a moniliform way with a small appendage, thin-walled;
content as on lamellae sides; (2) as on lamellae sides.
Lamellae edges sterile, when older elements can con-
tain brown pigments; marginal cells (15)19.126.1
33.1(36) × (4)4.75.66.5(7) μm, undifferentiated,
cylindrical to narrowly clavate, thin-walled. Pileipellis
orthochromatic in Cresyl Blue, 175400 μm deep, not
sharply delimited from trama, gradually passing; sub-
pellis not delimited from suprapellis; hyphae 36μm
wide near trama, dense near surface and near trama,
irregularly oriented, more parallel and horizontal near
trama and surface, intricate everywhere, pigmented
near surface only, with no distinct gelatinous coating.
Acid-resistant incrustations absent. Hyphal termina-
tions near the pileus margin long, with multiple septa,
flexuous, thin-walled, filled with irregular refractive
bodies containing brown pigments; terminal cells
(41)51.470.289.0(125) × (3)4.35.46.5(8) μm,
narrowly cylindrical, on average apically constricted to
3.5 μm; subterminal cells and the cells below similar
in length or slightly shorter, similar in width or grad-
ually slightly wider, subterminal cells and cells below
sometimes branched. Hyphal terminations near the
pileus centre similar, terminal cells usually shorter
(28)40.859.878.8(106) × (3)4.05.06.0(7) μm,
subterminal cells and cells below not branching.
Pileocystidia hard to find; near the pileus margin
widely dispersed to rare, mostly 1-celled, but up to 3-
celled, long, terminal cells (73)79.1110.6142.1(
145) × 4.97.29.5(11) μm, cylindrical, flexuose, apically
mostly with 23 eccentric appendages or obtuse, content as
in hymenial cystidia or more granulose, without reaction in
sulfovanillin; near the pileus centre widely dispersed, 1-
celled, (63)79.4106.7134.0(151) × (4)5.06.47.8(
8) μm, cylindrical to slightly subulate, apically with double
constriction or 12 eccentrical appendages, content as near
pileus margin. Oleiferous hyphae containing brown pig-
ments and cystidioid hyphae present in the trama.
Ecology: Growing with Quercus robur, Pinus sylvestris,
and Betula pendula.
Distribution: Known from Estonia, Germany, Slovakia,
Spain, and Sweden.
Additional material studied:Germany:Brandenburg,
Landkreis Oder-Spree, near Helenesee Frankfurt-Oder,
Markendorfer Forst, MTB 3752/2 (Müllrose), N 52.256386
E 14.46049, lichen-pine (Pinus sylvestris) forest with inter-
spersed birch trees (Betula pendula)onanunpaved
forest path, on sandy soil, 19 Oct. 2008, F. Hampe &
I. Kindermann, FH 2008 ST01 (hb. Felix Hampe).
Notes: The range of the spore size is large within this
species. This results from the sporesizedifferencebetween
the collections. The holotype (SAV F-3558) has smaller
spores and lower Q-value than collection FH 2008 ST01.
The variability in the shape of the hymenial cystidia is also
due to the difference between the collections with the holo-
type having cystidia of type 1 and collection FH 2008 ST01
having cystidia of type 2 (types referring to (1) and (2) in
the description). Therefore, based on morphology, we
could hypothesize that these collections represent different
species. Nevertheless, we treat them here as the same
species because phylogenetically there is no support for the
hypothesis of two different species. All markers used in this
study place these two collections together as the same
species. Measurements are given for each collection
separately in Supplementary Material 1. Of course, more
collections and more micromorphological study are
needed in order to understand this intraspecific variation.
The UNITE species hypothesis at 0.5% corresponding to
our concept of R. ambusta is based on a locked sequence
and after this publication we will propose to change the
reference sequence to the holotype of the species.
Russula nigrifacta De Lange & Adamčík, sp. nov.
(Figs. 3a-b, 11,12 and 13).
MycoBank: MB839081.
Etymology: Named after the strong blackening of the
basidiomata.
Diagnosis: Differs from the other species of the
Russula albonigra complex by the lack of both appendages
and bifurcations on the pileocystidia.
Type:Italy: Tuscany, Province of Livorno, Piombino,
with Quercus ilex and Quercus suber, 9 Nov. 2016, R. De
Lange, RDL 16044 (GENT holotype).
De Lange et al. IMA Fungus (2021) 12:20 Page 16 of 31
Description:Pileus large, 75105 mm diam, planocon-
vex to applanate, centrally depressed to umbilicate, be-
coming more infundibuliform when older; margin slightly
inflexed when young, straight when mature, smooth; pi-
leus surface smooth, sometimes slightly cracked at the
margin, dry, dull to somewhat viscid when wet; ivory to
cream, sand coloured, yellowish white (4A2, 5A3, 5B3)
with darker spots of pale brownish/greyish orange, yellow-
ish brown to light brown, dark brown (5B6, 5E4, 5E8,
5F4). Lamellae narrow, segmentiform to subventri-
cose, 24 mm deep, adnate to subdecurrent; white to
yellowish white, rarely with faintly blueish shine,
blackening with age; with numerous lamellulae of different
lengths without clear regular pattern, rarely locally anasto-
mosing; dense (69L+89l/cm at mid-radius); edges
even, concolorous, blackening with age. Stipe 3060 × 19
30 mm, cylindrical, firm and fleshy, smooth; white, becom-
ing more orange brown with age; solid inside. Context ca.
67 mm thick at mid-radius, firm, white, blackening with-
out reddening, surface of pileus and stipe also slightly red-
dening before blackening; turning greenish with FeSO
4
,
yellowish with KOH, immediately dark blue with guaiac
(strong reaction, +++); taste mild to slightly refreshing;
odour fruity, sweet. Spore print white (Ia).
Fig. 11 Russula nigrifacta (RDL 16028, RDL 16044/2, RDL 16063, SAV F-2418), hymenium. aBasidia. bMarginal cells. cBasidiospores. dCystidia
near lamellae edges. eCystidia on lamellae sides. Bar = 10 μm, except for c 5 μm
De Lange et al. IMA Fungus (2021) 12:20 Page 17 of 31
Basidiospores (6.7)7.58.08.5(9.5) × (5.0)5.76.0
6.3(7.0) μm, broadly ellipsoid to ellipsoid, Q =
(1.15)1.251.341.43(1.56); ornamentation of low, dense
[(5)710(11) in a 3 μm diam circle] amyloid warts, 0.2
0.4 μm high, subreticulate, abundantly fused into chains
[(0)36(8) fusions in a 3 μm diam circle] and also con-
nected by short, fine line connections [03(4) in a
3μm diam circle]; suprahilar spot small, not amyloi d.
Basidia (50)57.062.868.5(79) × (8)8.79.6
10.5(11) μm, narrowly clavate, 4-spored. Hymeni al cystidia
(57)62.682.6102.6(128) × (7)7.78.910.1(15) μm,
cylindrical to narrowly fusiform to narrowly clavate, some-
times slightly flexuose, apically obtuse or with small ap-
pendage, thin-walled; with heteromorphous, oily content,
fragmented in multiple crystalline-like masses, without
clear reaction in sulfovanillin; near the lamellae edges,
(37)53.069.485.8(119) × (7)8.09.110.2(11) μm,
cylindrical to narrowly fusiform, sometimes narrowly
clavate, often slightly flexuose, apically obtuse with small
appendage or mucronate to tapering towards the top,
thin-walled; content as on lamellae sides. Lamellae edges
sterile, when older elements can contain brown pigments;
Fig. 12 Russula nigrifacta (RDL 16028, RDL 16044/2, RDL 16063, SAV F-2418, SAV F-2419), hyphal terminations of the pileipellis. aNear the
pileus margin. bNear the pileus centre. Bar = 10 μm
De Lange et al. IMA Fungus (2021) 12:20 Page 18 of 31
marginal cells (15)17.722.226.7(30) × (4)4.65.9
7.2(10) μm, poorly differentiated, cylindrical to narrowly
clavate, flexuose, thin-walled. Pileipellis orthochromatic in
Cresyl Blue, 90136 μm deep, not sharply delimited from
trama and not gradually passing, intermediate; subpellis
not well delimited from suprapellis; hyphae 2.57μmwide
near trama, dense near surface and near trama, irregularly
oriented, more parallel and horizontal near trama, pigmen-
ted throughout the pileipellis, with some gelatinous coating.
Acid-resistant incrustations absent. Hyphal terminations
near the pileus margin long, slender, with multiple septa,
flexuous, thin-walled, filled with irregular refractive bodies
containing brown pigments; terminal cells (50)63.583.5
103.5(133) × (3)4.04.95.8(7) μm, narrowly cylin-
drical; subterminal cells and the cells below similar in
length and width, subterminal cells never branched.
Hyphal terminations near the pileus centre similar;
terminal cells (42)53.179.6106.1(135) × (3)3.4
4.65.8(8) μm, sometimes more subulate, subter-
minal cells and cells below shorter, subterminal cells
rarely branched. Pileocystidia near the pileus margin
dispersed, 14 celled, very long, terminal cells
Fig. 13 Russula nigrifacta (RDL 16028, RDL 16044/2, RDL 16063, SAV F-2418, SAV- F-2419), pileocystidia. aNear the pileus margin. bNear the
pileus centre. Bar = 10 μm
De Lange et al. IMA Fungus (2021) 12:20 Page 19 of 31
(41)83.4119.5155.6 (235) × (5)5.86.77.6(9) μm, cy-
lindrical, flexuose, apically obtuse or with slight constriction,
with oily granulose content, without clear reaction in sulfova-
nillin; near the pileus centre dispersed, 12 celled, similar in
shape and content or with less and more oily guttulate con-
tent, terminal cells (64)80.9123.0165.1(220) × (4)5.2
6.06.8(7) μm. Oleiferous hyphae containing brown pig-
ments and cystidioid hyphae present in the trama.
Ecology: Growing with Mediterranean oaks (Quercus
ilex and Quercus suber) in Italy, in Slovakia with Quercus
robur, and Carpinus betulus.
Distribution: Known from Estonia, Italy, and Slovakia.
Additional material studied:Italy: Tuscany, Province of
Livorno, Piombino, with Quercus ilex and Quercus suber,7
Nov. 2016, R. De Lange,RDL16028 (GENT); Tuscany, Prov-
ince of Livorno, Piombino, with Quercus ilex and Quercus
suber, 11 Nov. 2016, R. De Lange,RDL16063 (GENT).
Slovakia:Obyce,forestNEofthevillage,withQuercus and
Carpinus, 24 July 2008, S. Adamčík, (SAV F-2418); Prenčov,
Horné Majere, with Quercus and Carpinus, 22 July 2008,
S. Adamčík, (SAV F-2419); T e pličky, with Quercus
and Carpinus, 4 July 2009, S. Adamčík, (SAV F-3006);
Bohunický Roháč, forest close to the nature reserve, with
Quercus,8Sept.2006,S. Adamčík, (SAV F-1501).
Fig. 14 Russula ustulata (AV 16019), hymenium. aBasidia. bMarginal cells. cBasidiospores. dCystidia near lamellae edges. eCystidia on lamellae
sides. Bar = 10 μm, except for c 5 μm
De Lange et al. IMA Fungus (2021) 12:20 Page 20 of 31
Notes: Besides the diagnostic features mentioned in the diag-
nosis, R. nigrifacta is characterised by its higher spore orna-
mentation (incomplete reticulum) and the thin pileipellis.
These features are shared with R.albonigra,whichis
easily differentiated from the other species in the
complex by the oily guttulate content of the cystidia.
Furthermore, our data suggests that R.nigrifacta is associ-
ated with Quercus spp. (and possibly also with Carpinus
betulus) in thermophilous habitats (thermophilous oak
forests and Mediterranean oak forests).
Russula ustulata De Lange & Verbeken, sp. nov. (Figs.
3h, 14 and 15).
MycoBank: MB839082.
Etymology: Refers to the appearance of the basidio-
mata, which look like they were burnt.
Diagnosis: Differs from the other species of the Russula albo-
nigra complex by the absence (or rareness) of pileocystidia.
Type:Norway: NT Steinkjer, Kvamsfjellet, North of Lyst-
jörna, Austerolsenget, alt. 137.5 m, N64°1247E11°4909,
20 Aug. 2016, A. Verbeken,AV16019 (GENT holotype).
Description:Pileus large, 101112 mm diam, planoconvex
to applanate, centrally depressed to umbilicate to infundibu-
liform, widely V-shaped; margin straight, smooth; pileus sur-
face smooth, glabrous, dry, shiny; very dark blackish brown
to dark grey-black (7F24) without lighter brown tints, uni-
form. Lamellae narrow, segmentiform to subventricose, 45
mm deep, adnate to subdecurrent; completely white to
Fig. 15 Russula ustulata (AV 16019), pileipellis. aHyphal terminations near the pileus margin. bHyphal terminations near the pileus centre.
cPileocystidia near the pileus margin. Bar = 10 μm
De Lange et al. IMA Fungus (2021) 12:20 Page 21 of 31
yellowish white, quickly blackening; with numerous
lamellulae of different lengths, locally anastomosing; mod-
erately distant (69L+34 l/cm at mid-radius) to distant
(sometimes almost as in R. nigricans); edges even, papery
thin, concolorous, quickly blackening with age. Stipe 45
65 × 2530 mm, cylindrical, firm and fleshy, smooth to
irregular surface, dry; white but rapidly turning dark grey,
black (stays whitish only at the top); solid inside. Context
ca. 59 mm thick at mid-radius, firm, white, staining grey,
then brownish black, mostly blackening without redden-
ing but a slight pink tinge can be present; turning greenish
with FeSO
4
; taste mild but not agreeable, musty, but
slightly menthol-like in the gills. Spore print white (Ia).
Basidiospores (7.4)8.18.58.9(9.3) × (5.7)6.06.3
6.6(6.9) μm, broadly ellipsoid to ellipsoid, Q =
(1.12)1.261.361.46(1.48); ornamentation of very
low, very dense [(9)1015(17) in a 3 μm diam circle]
amyloid warts, to 0.2 μm high, reticulate, abundantly
fused into chains [37(10) fusions in a 3 μm diam
circle], abundantly connected by short, fine line connec-
tions [(6)711(13) in a 3 μm diam circle]; suprahilar
spot large, not amyloid. Basidia (53)58.967.375.7(
83) × (8)8.49.210.0(11) μm, narrowly clavate to cy-
lindrical, 4-spored. Hymenial cystidia (78)88.7106.6
124.5(153) × (7)8.09.110.2(12) μm, cylindrical to
narrowly fusiform, apically obtuse with small appendage
or slightly tapering towards the top, thin-walled; with
heteromorphous, oily content, fragmented in multiple
masses to needle-like crystalline, without reaction in sulfo-
vanillin; near the lamellae edges, (60)70.082.995.8(
111) × (7)8.19.510.9(14) μm, narrowly fusiform to
narrowly conical, slightly flexuose, apically obtuse, some-
times with small appendage or tapering towards the top,
thin-walled; content as on lamellae sides, often containing
brown pigments. Lamellae edges sterile, when older ele-
ments can contain brown pigments; marginal cells
(14)19.826.533.2(39) × 4.96.37.7(10) μm, poorly
differentiated, cylindrical to narrowly clavate to fusiform,
flexuose, thin-walled. Pileipellis orthochromatic in Cresyl
Blue, 250300 μm deep, not sharply delimited, gradually
passing; subpellis not delimited from suprapellis; hyphae
37μm wide near trama, dense near surface and near
trama, loose in intermediate zone, irregularly oriented,
pigmented throughout the pileipellis, some gelatinous
coating can be present deeper in the pileipellis. Acid-resist-
ant incrustations absent. Hyphal terminations near the
pileus margin long, with multiple septa, flexuous, thin-
walled, filled with irregular refractive bodies containing
brown pigments; terminal cells (29)36.053.671.1(
100) × (4)4.96.07.1(9) μm, narrowly cylindrical to
subulate, on average apically constricted to 4.5 μm; subter-
minal cells and the cells below shorter and gradually
wider, subterminal cells never branched, cells below occa-
sionally branched. Hyphal terminations near the pileus
centre slightly slender and apically less attenuated;
terminal cells slightly longer, (31)43.359.074.7(
89) × (3)4.05.16.2(8) μm, subterminal cells and cells
below more of similar size as terminal cells, subterminal
cells rarely branched. Pileocystidia near the pileus margin
extremely rare (only 5 observed), inconspicuous, hardly
distinguishable, 58.279.6101.0(115) × (6)6.37.0
7.7(8) μm, cylindrical to narrowly clavate, apically ob-
tuse, content as hymenial cystidia but very little, near the
pileus centre absent. Oleiferous hyphae containing brown
pigments present in the trama, cystidioid hyphae absent.
Ecology: Growing with Picea abies, and Pinus sylvestris.
Distribution: Known from the Czech Republic, Estonia,
Finland, Italy, Norway, and the Russian Federation.
Additional material studied:Italy: Langhestel, with Picea and
Pinus sylvestris, 25 Sept. 1997, S. Adamčík, (SAV F-2610).
Czech Republic: South Bohemia, Český Krumlov district,
Malonty, with Picea and Pinus sylvestris,togetherwithTricholoma
matsutake, 2 Sept. 2014, J. Borovička, (PRM 924452).
Notes: Our data suggests that Russula ustulata has a
specific ecology, different from the other species in the
complex. It is associated with coniferous trees in boreal
forests or mountain habitats.
Russula sp. 1 (Figs. 16,17 and 18).
Description:Pileus large, planoconvex to applanate,
centrally depressed to umbilicate to infundibuliform;
margin straight, smooth; pileus surface smooth, dry, dull;
light brown to greyish brown, dark brown, with paler
white to sand coloured patches. Lamellae narrow, seg-
mentiform to subventricose, adnate to subdecurrent;
completely white to yellowish white, quickly blackening;
with numerous lamellulae of different lengths; moder-
ately distant; edges even, concolorous, quickly blacken-
ing with age. Stipe cylindrical, firm and fleshy, smooth to
irregular surface, dry; white but turning orange brown to
dark grey, black; solid inside. Context firm, white, blacken-
ing without reddening; taste mild. Spore print white (Ia).
Basidiospores (7.4)7.67.98.2(8.4) × (6.0)6.16.3
6.5(6.6) μm, broadly ellipsoid, Q = (1.18)1.221.26
1.30(1.33); ornamentation of very low, very dense
[(8)915(16) in a 3 μm diam circle] amyloid warts, to
0.3 μm high, reticulate, abundantly fused into chains
[(1)27(11) fusions in a 3 μm diam circle],
abundantly connected by short, fine line connections
[(5)713(15) in a 3 μm diam circle]; suprahilar spot
medium-sized, not amyloid. Basidia (54)56.760.965.1(
69) × (9)9.49.910.4(11) μm, narrowly clavate, 4-spored.
Hymenial cystidia,64.083.6103.2(130) × (7)7.98.7
9.5(10) μm, cylindrical to narrowly fusiform, often slightly
moniliform and flexuose, apically obtuse or with double con-
striction or small appendage, thin-walled; with heteromorph-
ous, oily content, fragmented in multiple crystalline-like
masses, without reaction in sulfovanillin; near the lamellae
De Lange et al. IMA Fungus (2021) 12:20 Page 22 of 31
edges, (53)56.262.368.4(76) × (6)6.97.88.7(9) μm,
cylindrical to narrowly fusiform, often slightly moniliform
and flexuose, apically obtuse or with small appendage, thin-
walled; content as on lamellae sides. Lamellae edges sterile,
elements containing brown pigment when older; marginal
cells (15)16.321.526.7(30) × (5)5.36.67.9(9) μm,
undifferentiated, cylindrical to narrowly clavate, thin-walled.
Pileipellis orthochromatic in Cresyl Blue, 200275 μm deep,
not sharply delimited from trama and not gradually passing,
intermediate; subpellis not delimited from suprapellis; hy-
phae 35μmwideneartrama,moredensenearsurfaceand
near trama, irregularly oriented, more parallel and horizontal
near trama, pigmented only in the upper part of the pileipel-
lis, with some gelatinous coating. Acid-resistant incrustations
absent. Hyphal terminations near the pileus margin long,
with multiple septa, scarcely branched at the bases, flexuous,
thin-walled, filled with irregular refractive bodies containing
brown pigments; terminal cells (39)47.661.475.2(86) ×
5.06.07.0(8) μm, narrowly cylindrical to slightly subulate
or narrowly fusiform, on average apically constricted to
4.5 μm; subterminal cells and the cells below similar in length
and width or slightly wider, subterminal cells never branched.
Hyphal terminations near the pileus centre slightly wider and
apically more attenuated, containing inflated cells, more
Fig. 16 Russula sp. 1 (RW 1975), hymenium. aBasidia. bMarginal cells. cBasidiospores. dCystidia near lamellae edges. eCystidia on lamellae
sides. Bar = 10 μm, except for c 5 μm
De Lange et al. IMA Fungus (2021) 12:20 Page 23 of 31
Fig. 18 Russula sp. 1 (RW 1975), pileocystidia. aNear the pileus
margin. bNear the pileus centre. Bar = 10 μm
Fig. 17 Russula sp. 1 (RW 1975), hyphal terminations of the pileipellis.
aNear the pileus margin. bNear the pileus centre. Bar = 10 μm
De Lange et al. IMA Fungus (2021) 12:20 Page 24 of 31
often branched at the bases; terminal cells (28)46.370.5
94.7(130) × (3)3.95.26.5(8) μm, subterminal cells
rarely branched. Pileocystidia near the pileus margin numer-
ous, 1 celled, rarely 2 celled, extremely long, terminal cells
98more than 320 × (8)8.29.110.0 μm, cylindrical, flex-
uose, apically obtuse or with slight constriction, sometimes
bifurcating, content as in hymenial cystidia or more granu-
lose, without reaction in sulfovanillin; near the pileus centre
numerous, 12 celled, generally shorter but still very long,
terminal cells 56.6117.4178.2(298) × (5)6.18.410.7(
14) μm, similar in shape and content, rarely with up to 3
eccentric appendages. Oleiferous hyphae containing brown
pigments and cystidioid hyphae present in the trama.
Ecology: Growing with Mediterranean cork oak (Quercus
suber).
Distribution: Known from Italy (Sardinia).
Specimen examined:Italy: Sardinia, Tempio Pausania,
road SS133, between 3 and 4 km from centre, with Quercus
suber, 1 Nov. 2000, R. Walleyn,RW1975(GENT).
Notes: Although there is molecular and morphological
support that this collection represents a species different
from the other species in the Russula albonigra complex,
the authors chose to not formally describe this
Key to the European species of Russula subgen. Compactae
1 Height of spore ornamentation not exceeding 0.5 μm .......................................................... 2
Height of spore ornamentation exceeding 0.5 μm ....................................... roseonigra
2 (1) Content of hymenial cystidia not reacting in sulfovanillin ..................................................... 3
Content of hymenial cystidia at least greying in sulfovanillin ...................................... 8
3 (2) Basidiomata sturdy and fleshy; lamellae thick and very distant; context quickly and strongly reddening
before Lamellae thin and moderately distant; context not reddening before blackening or only slightly at the
surface; pileocystidia often exceeding 90 μm ................................ 4
4 (3) Content of all cystidia oily guttulate ........................................................................ albonigra
Content of cystidia not oily guttulate .......................................................................... 5
5 (4) Pileocystidia absent or extremely rare and only near the pileus margin ..................... ustulata
Pileocystidia at least widely dispersed and found throughout the whole surface of the pileus
......................................................................................................................... 6
6 (5) Pileocystidia never with appendages or bifurcations ............................................... nigrifacta
Pileocystidia (at least some) containing appendages or bifurcations ........................... 7
7 (6) Pileocystidia numerous, extremely long (some exceeding 200 μm), with both appendages and bifurcations
present; hyphal terminations of the pileipellis with inflated cells ................ sp. 1
Pileocystidia widely dispersed, never exceeding 160 μm, lacking bifurcations; hyphal terminations of the
pileipellis without inflated cells ....................................... ambusta
8 (2) Taste mild ............................................................................................................................ 9
Taste, at least in the lamellae, noticeably acrid......................................................... 10
9 (8) Context not clearly reddening but quickly and strongly blackening, sometimes with a slightmenthol-like
taste Context turning slowly pale pink before greying (but almost simultaneously, and without strong
blackening); smell of old wine barrels; pileocystidia present; spore ornamentation not exceeding 0.2
μm................................................................ adusta
10 (8) Hyphal terminations of the pileipellis consisting of shorter, broad, swollen cells ................ 11
Hyphal terminations of the pileipellis consisting of longer, slender cells ................... 12
11 (10) Context clearly reddening before turning grey-black .............................................. densifolia
Context not clearly reddening, first greying then blackening....................... densissima
12 (10) Context quickly and strongly blackening; lamellae with a distinct pinkish shine; pileocystidiaabsent;
hyphal terminations Context not so strongly and quickly blackening, more greying or slowly blackening;
pileocystidia present ................................................................................................ 13
13 (12) Context strongly reddening, then greying; taste burning acrid, in the flesh as well as in the lamellae
acrifolia Context not clearly reddening, slowly blackening; taste acrid, but not burning and more prominent
in the lamellae than in the context..................................... fuliginosa
De Lange et al. IMA Fungus (2021) 12:20 Page 25 of 31
species here as the description is based on a single
collection only.
DISCUSSION
Our study recognises five species within the traditional
concept of Russula albonigra and this name was the only
available at the species rank. Before new names could be
assigned to undescribed species, identification of the old
name proofed to be challenging, especially because it
was already used in the early Friesian period and its ori-
ginal description (Krombholz 1845) does not meet
current morphological standards and does not provide
sufficient detail. The species clade identified in this study
as R. albonigra has the best match with the probable
geographic and ecological origin of the species described
by Krombholz.
The traditional morphological concept of R. albonigra
(Romagnesi 1967; Sarnari 1998; Kibby 2001) more or
less agrees with our observations of the species complex.
We think that in the field, for the preliminary identifica-
tion of species to the R. albonigra complex, the following
characters are most helpful: a strong and fast blackening
of the surface resulting in a distinct contrast of wounded
compared to untouched areas, the relatively sturdy and
thick-fleshed basidiomata, a mild or refreshing taste of
the context, moderately distant and strongly blackening
lamellae and a dry, usually dull and not viscid pileus cu-
ticle. However, macroscopically the species within the R.
albonigra species complex are very alike and cannot be
distinguished unambiguously. Nonetheless, we observed
that the reaction to FeSO
4
could be interesting to differ-
entiate some of the species. While R.albonigra has an
orange reaction to FeSO
4
, both R.nigrifacta and R.ustu-
lata have a greenish reaction. As this data is missing for
R.ambusta and R. sp. 1, it is important to pay attention
to this character in future collections. There are some
interesting observations questioning the traditional char-
acteristics used to define the R.albonigra complex. First
of all, it seems that the absolute lack of any reddening is
not a reliable feature, because it can be weak and easily
overseen or vanishing quickly due to the strong and
quick blackening. At least three of the species within the
R.albonigra complex (R.albonigra, R.nigrifacta, and R.
ustulata) comprise a collection where some weak red-
dening is observed at the surface or even of the context.
Some variability about the reddening reaction was also
noted by Romagnesi, who recognised Russula albonigra
f. pseudonigricans with an intense reddening context
(Romagnesi 1962; Romagnesi 1967). Attempts to get a
sequence from the type material failed. The holotype of
this form is in a bad condition which does not allow
good microscopic observations. Furthermore, a micro-
scopic study of a paratype suggested that holotype and
paratype did not represent the same species. We suggest
for now, until molecular data becomes available, not to
draw any conclusions about the identity of R.albonigra
f. pseudonigricans or its classification within the R.albo-
nigra complex. Moreover, names described in our study
have priority at species rank over any future combin-
ation of the f. pseudonigricans epithet (Art. 11.2 of the
ICNafp). Although we believe that the reddening reac-
tion of the context can be used as a diagnostic between
species on opposite sides of the spectrum (i.e. species
with a strong reddening reaction versus species without
a clear reaction) some caution is needed.
Another traditional morphological character to define
R. albonigra was the characteristic menthol taste in the
lamellae. This does not seem to be a stable character be-
cause it was not observed in all collections and it can
also depend on the subjective opinion of an individual
person. Furthermore, this menthol-refreshing taste is
also noted to possibly be present in R.atramentosa by
Sarnari (1998).
We found the lack of a reaction of the cystidial con-
tent to sulfovanillin to be a good synapomorphic charac-
ter to define the R. albonigra complex. Russula nigricans
is the only species outside this complex also showing no
clear reaction of the cystidial content to sulfovanillin.
But the latter species can easily be distinguished from
the R.albonigra complex by its thick and very distant la-
mellae, the strong reddening of the context and its pileo-
cystidia that are much shorter (never exceeding 90 μm).
Our conclusions about the delimitation of the species
complex are only based on observations of European
taxa of R. subgen. Compactae.
The second challenge of this study was to define mor-
phological differences among the species of the R. albo-
nigra complex defined by phylogenetic analyses. Due to
the low morphological variability we could consider the
species within the R.albonigra complex pseudocryptic
species (i.e. species with a morphological resemblance
that seems indistinguishable at first, but can be distin-
guished when using the appropriate characters; Delgat
et al. 2019). This is a phenomenon that is widely distrib-
uted within the Russulaceae, especially in the genus Lac-
tifluus (Stubbe et al. 2010, Van de Putte et al. 2010, Van
de Putte 2012, De Crop et al. 2014, Van de Putte et al.
2016, Delgat et al. 2017, De Lange et al. 2018, Delgat
et al. 2019), but also within the genus Russula (Adamčík
et al. 2016a; Adamčík et al. 2016b; Caboňet al. 2019).
The most striking microscopical differences between the
species in the R. albonigra complex are the higher spore
ornamentation of R.nigrifacta and R.albonigra com-
pared to the other species within the complex. Russula
ustulata and R. sp. 1 have an almost complete and
denser reticulum than the incomplete reticulum of R.
nigrifacta and R.albonigra. The ornamentation of the
spores in R.ambusta is intermediate in reticulation and
De Lange et al. IMA Fungus (2021) 12:20 Page 26 of 31
density. R.albonigra is distinguishable by the unique oily
guttulate content of all cystidia. R.nigrifacta typically
lacks appendages and bifurcations on the pileocystidia
whereas these are present in R.albonigra and R. sp. 1.
Russula ambusta lacks bifurcations but appendages are
present. The most striking feature of R.ustulata is the
absence (or rareness) of pileocystidia, whereas R. sp. 1
has very long and numerous pileocystidia. The thickness
of the pileipellis is also an interesting character. R.albo-
nigra has the thinnest pileipellis followed by R.nigri-
facta, the other species in the complex have a much
thicker pileipellis. The presence of inflated subterminal
cells in the hyphal terminations of the pileipellis centre
is typical for R. sp. 1 and not observed in the other spe-
cies of the complex.
Collections used in this study often do not have pre-
cise ecological details to define ecological niches and
host tree preferences. However, habitat type and geo-
graphical data suggest biological relevance to recognise
closely related species (Ryberg 2015). Russula ambusta,
R. nigrifacta, and R. ustulata are closely related but seem
to inhabit ecologically different niches. Russula nigri-
facta occurs both with Mediterranean oaks (Quercus ilex
and Quercus suber) and Quercus robur and Carpinus
betulus in thermophilous oak forests. Possibly, Russula
sp. 1 has a similar ecology,it is only known from a single
collection associated with Mediterranean oak (Quercus
suber). Russula ustulata is up to now only known from
boreal or mountain habitats, associated with coniferous
trees (Picea sp., Pinus sp.). Russula ambusta and R.albo-
nigra seem to have a similar ecology and are associated
with a variety of trees in temperate to montane forest
types. Russula ambusta was collected with Quercus
robur,Pinus sylvestris, and Betula pendula;Russula
albonigra with Fagus sylvatica,Abies alba,Picea abies,
and Carpinus betulus.
Our study suggests that species within the Nigricanti-
nae clade have a limited area of distribution, unlike what
is often believed. All North American collections re-
trieved from GenBank and placed in the R. albonigra
complex are not clustered with the European ones and
probably represent different species (Fig. 2). The re-
trieved ITS data did not confirm that the distribution of
R. albonigra is not transcontinental (Singer 1958; Hesler
1961; Shaffer 1962; Kibby and Fatto 1990; Thiers 1994),
but rather it supports the hypothesis that none of the
European taxa within R. subgen. Compactae are present
in the United States (Adamčík and Buyck 2014). The
North American collections represent at least two differ-
ent species with a high macromorphological resemblance
to R.albonigra and they may represent R. sordida and R.
subsordida, both having a weak or negative reaction of the
pileocystidia to sulfovanillin (Adamčík and Buyck 2014).
This study shows that the R. albonigra complex is also
represented in Asia by a still undescribed Chinese species.
A multi-locus phylogeny resulting in a strong sup-
port of the European species within the R.albonigra
complex, stimulated the detailed search for morpho-
logical differences between the species. The species
described in this study are defined by integrated tax-
onomy combining multi-locus molecular data with
detailed morphological and ecological data (i.e. distri-
bution, climate, and host data).
Our study demonstrated that all species within the R.
albonigra complex, supported by the strict genealogic
concordance and coalescent-based species delimitation,
are strictly distinguished at the threshold of 99.5%, that
corresponds to a distance of 0.5% when performing a
UNITE search. Even a distance of 1% results in only two
UNITE species hypotheses both covering multiple phylo-
genetic species within the complex.This shows that there
is a low genetic diversity of the ITS region between the
species within this complex. A possible explanation for
this low genetic diversity is that the species within
the R.albonigra complex are only relatively recently di-
verged from each other, which could explain the relatively
short branch lengths (Fig. 2) and the low morphological
variability. The occupation of new habitats and the adap-
tation to new hosts could have caused the radiation seen.
This study shows that ITS sequence similarity thresh-
olds of 97% commonly used in metabarcoding studies
(Pauvert et al. 2019) are not sufficient to differentiate the
phylogenetically defined species of the R.albonigra com-
plex. This observation is also made for other species
complexes within the genus Russula (Adamčík et al.
2016b). The species thresholds retrieved in this study
agree with the conclusions of testing global fungal data-
bases as training datasets, that predicted the optimal
identity thresholds to discriminate filamentous fungal
species as 99.6% or 99.3% for ITS (Vu et al. 2019;Vu
et al. 2020). Badotti et al. (2017) rank Russula as the
genus with only 38% probability of correct identification,
but the study also ranks it into the group for which ITS
is a good marker while another Russulaceae genus, Lac-
tarius, is placed in a group for which ITS is a poor
marker. We do not promote the use of a universal
threshold value, but rather emphasise the importance of
searching for the best threshold value, according to the
fungal group of interest.
Our UNITE species hypothesis threshold testing meets
both major problems highlighted by Kõljalg et al. (2013):
(1) the lack of an inclusive, reliable public reference data
set; and (2) the lack of means to refer to fungal species,
for which no scientific name is available, in a standard-
ized stable way. First, our singleton collection of R. sp.1
is not represented in UNITE, the other four European
species are represented by 220 sequences. Second, the
De Lange et al. IMA Fungus (2021) 12:20 Page 27 of 31
UNITE nomenclature refers to, in our opinion, an incor-
rect concept of R.albonigra and there is a lack of means
with which we can assign potential correct and valid
available names to the North American taxa.
As the result of a concerted effort to improve UNITE
annotations, Nilsson et al. (2014) designated, based on
relevant literature data, 1368 species hypothesis reference
sequences but also marked 363 sequences of compro-
mised quality. Sequences of compromised technical
quality often originate from amplicon sequencing of envir-
onmental samples and are biased by the sequencing tech-
nique, and some of them can be recognised by automated
chimera search or presence of IUPAC ambiguity codes
(Badotti et al. 2017; Nilsson et al. 2018). Our comparison
of distinguishing nucleotide positions (Additional file 1)
demonstrated that short ITS sequences usually do not
provide sufficient information for species identification,
i.e. sequences UDB0502905 and UDB031025 containing
only ITS2 region are undoubtedly identified to species,
but UDB0663165 and UDB0557800 provide dubious in-
formation. The sequence UDB065518 does not match any
species recognised in our tree due to low quality or pos-
sibly it represents a new taxon. Our study dealt with five
species and we identified four sequences of compromised
quality, this is a higher ratio of low quality sequences
detected compared to the above mentioned study of
Nilsson et al. (2014).
Hofstetter et al. (2019) highlight the main problems with
sequence-based identification of fungi. Besides poor taxon
coverage in public sequence databases, misidentification,
the use of wrong names and bad annotation of sequences,
remain a major problem. As sequence-based identification
becomes more and more routine and the standard ap-
proach for many (mainly ecological) studies, it is clear that
the annotation of public sequences urgently needs to be
improved. An important implementation is the recent
introduction of taxonomic hypothesis that communicate
SH with taxonomic identification (Kõljalg et al. 2020). It
allows the tracing of taxonomic concepts presented at
UNITE and to link them with data about other fungal
traits. Hofstetter et al. (2019) and Durkin et al. (2020)state
that taxonomy finds itself at the same risk of extinction as
the very species they are supposed to study and provide
some recommendations towards fungal taxonomists on
how to highlight the importance and improve taxonomic
work. Taxonomists should create a reliable taxonomic
framework that can be used by conservationists and ecolo-
gists for sequence based identification. Lucking et al.
(2020) provides a discussion on how to improve the qual-
ity of identification tools and states that these tools are
only as good as the reference data behind them. The
UNITE database favours third-party annotation by taxo-
nomic experts to improve the taxonomic annotation of
the sequence data (Nilsson et al. 2018). Additional file 3
provides our proposed correction to the annotation of the
sequences, with the immediate advantage that it can be
directly integrated into further studies.
CONCLUSION
Russula albonigra has always been seen as one of the
more easily recognizable species within R. subgen. Com-
pactae by its strongly and rapidly blackening reaction of
the context, without intermediate reddening, resulting in a
strong black-and-white contrast of wounded and un-
touched parts, and the menthol-cooling taste of the lamel-
lae. The species was also thought to cover a broad
ecological amplitude and large distribution area. However,
molecular analysis revealed that R.albonigra s. lat.repre-
sents a species complex consisting of at least five Euro-
pean, two North American, and one Chinese species. A
thorough morphological study shows that the characters
traditionally used are not always reliable to define the R.
albonigra complex, instead the lack of a reaction of the
cystidial content to sulfovanillin is proposed as a character
to improve delimitation of the R. albonigra complex. This
feature is only shared with R. nigricans which can be read-
ily distinguished by the more spaced lamellae. Our UNITE
species hypothesis threshold testing revealed perfect
phylogenetic species match at a sequence distance thresh-
old of 0.5% for the R.albonigra complex. UNITE species
hypothesis may be a powerful tool to improve knowledge
about distribution and ecology of studied species, but the
pitfalls include short and low quality sequences. For
phylogenetic analyses of the ITS region we recommend
the use of sequences with existing (not blank) UNITE spe-
cies hypotheses at the 0.5% threshold and sequences of full
length. Our observations can be applicable for the genus
Russula as a whole and to many other macrofungal
genera. The importance of looking for the best threshold
value according to the fungal group of interest is
emphasised.
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s43008-021-00064-0.
Additional file 1. Sequence similarity table. Table showing sequence
similarity at distinguishing nucleotide positions.
Additional file 2. Comparison table. Table with a detailed comparison
of microscopical characters.
Additional file 3. Proposed corrected sequence annotation. Table with
proposed corrected annotation of the taxonomic data of the public
sequences.
Acknowledgements
Helga Marxmüller is thanked for providing material from her personal
herbarium. Jesko Kleine is thanked for providing material from his personal
herbarium and checking the taxonomy. Miroslav Caboňis thanked for his
suggestions to improve the phylogenetic analysis. Geoffrey Kibby is thanked
De Lange et al. IMA Fungus (2021) 12:20 Page 28 of 31
for proofreading the manuscript as a native English speaker. The Entoloma
team, organizing the week of fieldwork in Steinkjer, Norway in August 2016,
is acknowledged for allowing Annemieke Verbeken to participate and for
supporting her in the fieldwork.
Authorscontributions
RDL provided and gathered collections/sequences to constitute the final
dataset, performed part of the molecular lab work, performed the molecular
analysis, performed the microscopical study, made the descriptions for the
new species and the microscopic plates and wrote the manuscript. SA
provided collections and contributed to the macroscopical descriptions,
aided the microscopical study and molecular analysis, was involved in the
setup of the concept and the design of the study, was a major contributor
in writing the manuscript. KA and PA performed part of the molecular lab
work, KA annotated and submitted all sequences to the GenBank database.
JB provided collections/sequences essential to the study (the collection used
to determine the clade representing R.albonigra) and contributed to the
macroscopical descriptions. LD provided collections and performed part of
the molecular lab work. FH provided collections, contributed to the
macroscopical descriptions and performed part of the molecular lab work.
AV is the promotor of the first author, provided collections and contributed
to the macroscopical descriptions, was involved in the setup of the concept
and the design of the study, was a major contributor in writing the
manuscript. All authors reviewed the manuscript. All authors read and
approved the final manuscript.
Funding
LD (grant BOFDOC2015007001) was funded by a scholarship of the Special
Research Fund and by the JEC (Journées européennes du Cortinaire) 2016
organisation during the collecting of specimens. The lab work of SA and KA
is funded by the Slovak national project APVV 150210. The overall work of
JB was supported by the Long-term Development Projects RVO67985831
and RVO61389005.
Availability of data and materials
All data generated or analysed during this study are included in this
published article [and its supplementary information files].
Declarations
Adherence to national and international regulations
We confirm adherence to any pertinent national or international legislation
or regulations that apply to the transfer of living biotic materials used in the
study between countries.
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Research Group Mycology, Department of Biology, Ghent University, K.L.
Ledeganckstraat 35, 9000 Ghent, Belgium.
2
Institute of Botany, Plant Science
and Biodiversity Center, Slovak Academy of Sciences, Dúbravská cesta 9, 845
23 Bratislava, Slovakia.
3
Institute of Forest Ecology Slovak Academy of
Sciences, Akademická 2, 949 01 Nitra, Slovakia.
4
Institute of Geology of the
Czech Academy of Sciences, Rozvojová 269, 165 00 Prague 6, Czech
Republic.
5
Nuclear Physics Institute of the Czech Academy of Sciences, Hlavní
130, 250 68 Husinec-Řež, Czech Republic.
6
Meise Botanic Garden, Research
Department, Nieuwelaan 38, 1860 Meise, Belgium.
Received: 26 November 2020 Accepted: 12 April 2021
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... Various studies have compared the species delimitation methods based on single-locus or multi-locus datasets and indicated that different methods often produce incongruent delimitation scenarios [22,46,[48][49][50][51][52][53]. To date, there is no similar study conducted on the delimitation of Chrysomxya rust species. ...
Article
Full-text available
Chrysomyxa rusts are fungal pathogens widely distributed in the Northern hemisphere, causing spruce needle and cone rust diseases, and they are responsible for significant economic losses in China. Taxonomic delimitation and precise species identification are difficult within this genus because some characters often overlap in several species. Adequate species delimitation, enhanced by the use of DNA-based methodologies, will help to establish well-supported species boundaries and enable the identification of cryptic species. Here, we explore the cryptic species diversity in the rust genus Chrysomyxa from China. Species delimitation analyses are conducted using a distance-based method (ABGD) and three tree-based methods (GMYC, bPTP, and mPTP) based on combined LSU and ITS sequences of over 60 specimens. Although there is some incongruence among species delimitation methods, two new species and three putative cryptic species are identified. The key to 20 Chrysomyxa species distributed in China is presented. These results suggest that a significant level of undiscovered cryptic diversity is likely to be found in Chrysomyxa from China. Future studies should consider multiple analytical methods when dealing with multi-locus datasets.
... Following the publication of the original article[1], we were notified of a few inconsistencies between the pdf version and the html version with regards to the Key to the European species of Russula subgen. Compactae (pg. ...